Ribosome biogenesis is one of the major biological pathways within the cell. Tightly controlled regulation of ribosomal RNA and protein synthesis and their assembly into ribosomes is critical to cellular fitness. Proper ribosome assembly is also important for the health of organisms, as at least one genetic disorder has been linked to a defect in ribosome biogenesis. Examination of expression of the unlinked, duplicated, RPS14A and RPS14B genes that encode ribosomal protein S14 (rpS14) in yeast has lead to an increased understanding of the control of eukaryotic ribosomal biogenesis. Expression of RPS14B is negatively regulated by binding of excess rpS14 to a specific stem-loop structure that encompasses both exonic and intronic sequences in RPS14B pre-mRNA. Interestingly the regulatory stem-loop structure in RPS14B pre-mRNA is mutually exclusive with a second structure that functions in vivo as an intronic enhancer, suggesting that there is an equilibrium between the enhancing and regulatory structures that is shifted towards the latter in the presence of excess rpS14. The mechanism of rpS14-mediated repression of RPS14B will be addressed by examining the expression of constructs with mutations that affect the formation of these structures in vivo and by examining RNA splicing and structure in vitro. These experiments are designed to allow a determination of whether regulation occurs at the level of pre-mRNA splicing or RNA turnover and to determine the roles of the regulatory and enhancer elements play in this regulation. In addition, the role of trans-acting factors in the RPS14B regulation will be explored.